We do not understand the mechanisms that bacteria use to grow. Rod shaped bacteria elongate by inserting new strands of glycans into their cell wall. These strands are cross-linked into a three-dimensional meshwork called peptidoglycan. This encapsulating structure not only defines the bacterial shape, it also protects them from the environment. Cells modulate how this structure is synthesized, cross-linked, or stiffened so that they can adapt to environmental changes, external stresses, or the presence of antibiotics. There are many different enzymes bacteria use to build and modify the cell wall, and while we know the reactions catalyzed by each enzyme, we do not know how these enzymes function in vivo: how does the emergent action of all synthetic enzymes create uniformly growing, perfectly shaped cells. This requires that the activity of each enzyme to be regulated in space so its activity is positioned at the proper locale. It is also not known how bacteria regulate enzymatic activity in time: how they modulate specific activities to respond to external stresses. The clinicl antibiotics that stop cell wall synthesis target the enzymes that build the cell wall. These antibiotics have been effective for many years, but the increasing spread of resistance to this class of antibiotics is limiting their utility. Therefore, by finding other points in the cell wall synthesis pathway, points that are fragile, we may find new proteins we can target to inhibit bacterial growth. Rather than the enzymes themselves, we will seek to target the mechanisms the cell uses to regulate the activity of the synthetic enzymes. It is believed this regulation occurs through proteins that associate with these enzymes, changing their location or activity. Genetic and biochemical studies are yielding a large list of possible regulating factors, but, this interaction map remains unclear: we do not know which components in this system interact, how these interactions modulate cell wall synthesis, much less when or where in the cell this interaction occurs. We have discovered that we can read out the associations and activity of cell wall synthesis proteins by quantitating their motions using in vivo single molecule tracking. We will use this approach to dissect cell wall synthesis, mapping out the protein-protein interactions as well as the communication that occurs between different enzymatic functions. First we build an interaction map by characterizing the dynamics of all known components. We then will test this map using genetic perturbations. To understand how information is communicated within this network, we will observe each proteins dynamics as we systematically deplete other genes. Finally, we will determine what the fragile points are in this system by studying native points of regulation: how bacteria control their rate of growth, and how they respond to antibiotic stresses.